Journal of Experimental Botany, Vol. 67, No. 13 pp. 3953–3964, 2016
doi:10.1093/jxb/erw089 Advance Access publication 8 March 2016
This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
RESEARCH PAPER
Blue light-dependent changes in loosely bound calcium in
Arabidopsis mesophyll cells: an X-ray microanalysis study
Justyna Łabuz1,2, Sławomir Samardakiewicz3, Paweł Hermanowicz1, Elżbieta Wyroba4, Maria Pilarska1,† and
Halina Gabryś1,*
1 Department of Plant Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland
Malopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland.
3 Laboratory of Electron and Confocal Microscopy, Faculty of Biology, Adam Mickiewicz University, Poznań, Poland.
4 Laboratory of Electron Microscopy, Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warsaw, Poland
2 * Correspondence: [email protected]
Present address: The Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Krakow, Poland.
† Received 29 July 2015; Accepted 11 February 2016
Editor: Markus Teige, University of Vienna Abstract
Calcium is involved in the signal transduction pathway from phototropins, the blue light photoreceptor kinases which
mediate chloroplast movements. The chloroplast accumulation response in low light is controlled by both phot1 and
phot2, while only phot2 is involved in avoidance movement induced by strong light. Phototropins elevate cytosolic
Ca2+ after activation by blue light. In higher plants, both types of chloroplast responses depend on Ca2+, and internal
calcium stores seem to be crucial for these processes. Yet, the calcium signatures generated after the perception of
blue light by phototropins are not well understood. To characterize the localization of calcium in Arabidopsis mesophyll
cells, loosely bound (exchangeable) Ca2+ was precipitated with potassium pyroantimonate and analyzed by transmission electron microscopy followed by energy-dispersive X-ray microanalysis. In dark-adapted wild-type Arabidopsis
leaves, calcium precipitates were observed at the cell wall, where they formed spherical structures. After strong blue
light irradiation, calcium at the apoplast prevailed, and bigger, multilayer precipitates were found. Spherical calcium
precipitates were also detected at the tonoplast. After red light treatment as a control, the precipitates at the cell wall
were smaller and less numerous. In the phot2 and phot1phot2 mutants, calcium patterns were different from those of
wild-type plants. In both mutants, no elevation of calcium after blue light treatment was observed at the cell periphery (including the cell wall and a fragment of cytoplasm). This result confirms the involvement of phototropin2 in the
regulation of Ca2+ homeostasis in mesophyll cells.
Key words: Arabidopsis thaliana, blue light, calcium signaling, chloroplast movements, mesophyll cells, phototropin2.
Introduction
Calcium ions are considered to be an extremely versatile secondary messenger, a key element of many responses to biotic
and abiotic factors (for a review, see Dodd et al., 2010). In
plants, specific ‘calcium signatures’ are generated, mainly
in the cytosol, but also in the nucleus, mitochondria, and
chloroplasts (Stael et al., 2012). The precise control of Ca2+
translocations between organelles and the apoplast generates
spatially and temporally distinct cytosolic calcium patterns
and thus produces stimulus-specific responses (McAinsh and
Pittman, 2009).
In the case of blue light signaling, calcium elevation follows
the activation of phototropins. Phototropins are UVA and
© The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/),
which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
3954 | Łabuz et al.
blue light photoreceptor kinases which mediate plant movements and rapid growth responses. The two phototropins of
Arabidopsis thaliana, phot1 and phot2, are characterized by
different light sensitivities, though they share highly redundant functions. They both control phototropism, leaf expansion, stomatal opening, and the chloroplast accumulation
response. Only phot1 mediates the inhibition of the hypocotyl
growth reaction, and only phot2 mediates chloroplast avoidance and dark positioning (for a review, see Christie, 2007).
Although both Arabidopsis phototropins are responsible for
Ca2+ mobilization after blue light treatment, differences in
the localization of cellular calcium signals depending on light
intensity, plant organ, and the phototropin itself have been
reported (Harada and Shimazaki, 2007).
In 10- to 16-day-old Arabidopsis seedlings, a pulse of very
strong blue light (10 s, 600 μmol m−2 s−1) resulted in a transient
increase in cytosolic calcium concentration, [Ca2+]cyt, which
diminished after treatment with lanthanum (La3+) ions (calcium channel inhibitors). In phot1 mutant seedlings, [Ca2+]cyt
was increased in darkness, but the elevation after blue light
treatment was half that of the wild type. This indicated the
role of phot1 in mobilizing Ca2+ from intercellular spaces
through cell membrane-localized channels (Baum et al.,
1999). In 3-day-old etiolated Arabidopsis wild-type seedlings,
an increase in calcium concentration was observed after a
pulse of blue light (10 s, 100 μmol m−2 s−1). This response was
inhibited by BAPTA (a Ca2+ chelator), similarly to the inhibition of hypocotyl growth, controlled by phot1 (Folta et al.,
2003). In 4-day-old etiolated Arabidopsis wild-type seedlings,
the illumination of hypocotyls and cotyledons with continuous blue light (25 μmol m−2 s−1) for 10 min caused a transient
increase in [Ca2+]cyt. In wild-type and phot2 mutant plants, the
biggest influx of calcium was detected in the third minute after
turning on the light. In the phot1 mutant, a residual effect was
present and, since it was not observed at all in the double
phot1phot2 mutant, calcium mobilization from the apoplast
was attributed to phot1 (Babourina et al., 2002). A potentialgated cation channel called PACC, a phototropin-activated
calcium-permeable channel, was identified in mesophyll protoplasts based on the observation of a Ca2+ flux after continuous illumination with blue light of 275 μmol m−2 s−1. This
process became saturated after 11–16 min of irradiation and
was inhibited by La3+ and protein kinase inhibitors. In the
phot1 mutant, the activity of this channel was greatly reduced
and it was not detected in the double phot1phot2 mutant
(Stoelzle et al., 2003).
In 3-week-old Arabidopsis rosette leaves, changes in
[Ca2+]cyt were observed after 10 s blue light pulses. In wildtype plants, an increase in calcium concentration occurred at
light intensities of 0.1–250 μmol m−2 s−1, phot1 being active
in the range of 0.1–50 μmol m−2 s−1 and phot2 in the range of
1–250 μmol m−2 s−1. Calcium channel inhibitors, Co2+, La3+,
and nifedipine, and calcium chelators, EGTA and BAPTA,
caused a significant reduction in the blue light response in
wild-type plants and phototropin mutants, indicating a mechanism of Ca2+ influx into the cytoplasm through membrane
channels regulated by both photoreceptors. On the other
hand, phospholipase C inhibitors neomycin and U-73122
inhibited the blue light increase in [Ca2+]cyt in wild-type and
phot1 mutant plants, but not in the phot2 mutant. Thus
phot2 was suggested to be responsible for the phospholipase
C-dependent release of Ca2+ from internal stores, such as the
vacuole or endoplasmic reticulum (ER) (Harada et al., 2003).
The role of calcium in the control of chloroplast movements was proposed long before the discovery of phototropins (for a review, see Banaś et al., 2012). The rotation of
the flat, ribbon-like chloroplast of the filamentous alga
Mougeotia was shown to depend on the presence of specialized membrane vesicles which contain calcium ions (Wagner
and Klein, 1981). However, other works using calcium inhibitors suggested that the disruptions in chloroplast rotation
resulted from toxic effects or disturbances in processes other
than the photosensory transduction chain (Schönbohm
et al., 1990). In Vallisneria gigantea, the movement of chloroplasts under red light correlated with cytoplasmic streaming
within the cell and was precisely regulated by Ca2+ concentration (Takagi and Nagai, 1983). In Physcomitrella patens
(Sato et al., 2003) and Adiantum capillus-veneris (Sato et al.,
2001), external calcium was involved in triggering chloroplast
movement induced by mechanical stimulation. Inhibitors of
calcium channels (La3+ and Ga3+) did not affect the photorelocation of chloroplasts in these species.
Two types of blue light-controlled chloroplast movements
have been characterized in higher plants. One is the accumulation response observed in Arabidopsis under low blue light
(0.08–4 μmol m−2 s−1). Chloroplasts move to cell walls lying
perpendicular to the direction of incident light. The other is the
avoidance response. Under strong blue light (>20 μmol m−2 s−1),
chloroplasts gather under walls parallel to the direction of incident light (Trojan and Gabryś, 1996; Sakai et al., 2001).
The involvement of calcium in the control of blue lightinduced chloroplast relocations was put forward on the basis
of inhibitor treatments in the aquatic angiosperm Lemna trisulca (Tlałka and Gabrys, 1993; Tlałka and Fricker, 1999).
A prolonged incubation (12 h) with EGTA was needed for a
partial inhibition of chloroplast avoidance. However, in tissues initially pre-treated with the A23187 ionophore, only 1 h
long EGTA incubation was sufficient to disturb chloroplast
movements. Similarly, A23187 pre-treatment followed by 1 h
incubation with lanthanum enhanced its inhibitory effect on
chloroplast relocations. These observations indicated the key
role of internal calcium stores in the control of chloroplast
movements (Tlałka and Gabrys, 1993). This hypothesis was
supported by strong inhibitory effects observed after 2 min
treatments with caffeine (which causes a release of calcium
from intracellular stores) and thapsigargin (a selective inhibitor of the ER Ca2+-ATPase). The incubation of Lemna
fronds with calcium channel inhibitors nifedipine (for 3 h)
and verapamil (for 1 h) also partially disturbed chloroplast
movements (Tlałka and Fricker, 1999).
In Nicotiana tabacum, the exogenous application of Ca2+
did not affect movements. However, the disturbance in calcium homeostasis by incubating the tissue with the A23187
ionophore proved detrimental to chloroplast relocations. In
tobacco, in contrast to duckweed, short-term treatment with
EGTA or TFP (trifluoperazine; an inhibitor of calmodulin)
Light-dependent calcium changes in Arabidopsis mesophyll | 3955
was sufficient to inhibit both chloroplast responses strongly.
This effect was at least partially caused by disorders in the
actin cytoskeleton, which is indispensable for movements. The
subsequent addition of Ca2+ partially restored the ability of
chloroplasts to move (Anielska-Mazur et al., 2009). A similar
movement reactivation after EGTA treatment was observed
in Adiantum (Kadota and Wada, 1992). Inhibition by TFP
implicated a calmodulin-dependent signal transduction pathway, consistent with previous findings on chloroplast rotation
control in Mougeotia (Wagner et al., 1984).
Indirect evidence for the role of calcium in the control of
chloroplast relocations in Arabidopsis came from a study
showing the involvement of phosphoinositides in the blue
light signaling pathway. The phospholipase C pathway was
suggested to take part in phot2 signaling, while the phosphatidylinositol kinases PI3K and PI4K were suggested to
control the accumulation response mediated by both phototropins. U73-122 (the phospholipase C inhibitor) as well as
wortmannin and LY294002 (two inhibitors of the PI3K pathway) suppressed the transient calcium elevation induced by
blue light in Arabidopsis leaves (Aggarwal et al., 2013a, b). In
line with that, the inhibitory effect of wortmannin on chloroplast movements in Nicotiana could be over-ridden by the
application of external Ca2+ (Anielska-Mazur et al., 2009).
Even though substantial evidence has been collected,
the role of calcium in phototropin-controlled physiological responses needs further investigation. Ca2+ fluxes have
been analyzed by aequorin luminescence (Baum et al., 1999;
Harada et al., 2003), ion-selective microelectrode (Babourina
et al., 2002), and patch-clamp methods (Stoelzle et al.,
2003). These show changes in calcium concentration mainly
as a function of time. Although indirect evidence has been
obtained using inhibitors, differences in the spatial distribution of Ca2+ within the cell and the relevant calcium storage
compartments remain to be specified. Studies on calcium in
Arabidopsis leaves (Harada et al., 2003) and mesophyll protoplasts (Stoelzle et al., 2003) determine light ranges in which
phot1 and phot2 affect Ca2+ concentrations; however, they
do not answer the question of how these changes correspond
to chloroplast movement signaling. Spatial and temporal
aspects of calcium release are important to understand how
specific calcium signatures are generated. In this work, Ca2+
precipitation with KPA (potassium pyroantimonate) and
transmission electron microscopy (TEM) followed by X-ray
microanalysis were employed to study changes in calcium distribution. The main aim was to determine the localization of
Ca2+ after blue light treatment in Arabidopsis mesophyll cells
in order to elucidate how calcium participates in directing
chloroplast movements. The intensity of blue light and the
duration of irradiation were chosen to investigate the phot2specific chloroplast avoidance response.
Materials and methods
Plant material and growth conditions
Seeds of Arabidopsis wild-type Columbia were obtained from
Nottingham Arabidopsis Stock Centre (Nottingham, UK). Mutant
seeds were the kind gifts of A.R. Cashmore, the Plant Science
Institute, Department of Biology, University of Pennsylvania,
Philadelphia, USA (phot2) and J. Jarillo, Instituto Nacional de
Investigación y Tecnología Agraria y Alimentaria, Madrid, Spain
(phot1phot2). Plants were grown in a growth chamber (Sanyo MLR
350H, Japan) with a 10 h/14 h light/dark cycle at 23 °C, with 80% relative humidity, and illuminated by fluorescent lamps (FL40SS.W/37,
Sanyo, Japan) with a photosynthetic photon flux density of
60–100 μmol m−2 s−1.
Tissue processing
Five-week-old plants were dark-adapted for at least 12 h. The fifth or
sixth rosette leaf was irradiated directly on the plant for 3 min with
blue light (LXHL-PR09, Ledium Ltd, Hungary) of 100 μmol m−2
s−1 to induce the phot2-mediated chloroplast avoidance response.
Control plants were kept untreated in darkness or were irradiated
for 3 min with equimolar red light (LXHL-PD09, Ledium Ltd),
which does not activate phototropins. Immediately after treatment,
leaves were cut into 2 mm strips and put directly into the fixative
solution. A suitable cutting margin was taken into account, so that
the wounded site did not lie adjacent to the cells used for analysis.
In order to minimize calcium elution, pyroantimonate precipitation was performed concomitantly with material fixing. Tissue sections were infiltrated with a syringe containing a fixative solution of
2% glutaraldehyde, 2% potassium pyroantimonate in a phosphate
buffer (100 mM KH2PO4/K2HPO4, pH 7.4) at 4 °C (according to
Tretyn et al., 1992; Musetti and Favali, 2003) and incubated on ice
for 2 h. All steps were performed in darkness, using only ‘safe’ green
light. After fixation, the material was washed four times in a chilled
phosphate buffer (100 mM KH2PO4/K2HPO4, pH 7.6, 3 × 10 min,
1 × 15 min) and subsequently stained with 1% osmium tetra-oxide
in a 100 mM phosphate buffer at 4 °C. After dehydration in an
acetone series (10, 30, 50, 70, 90, 96, 100%, 2 × 5 min), the material
was embedded in epoxy resin of low viscosity (Spurr, 1969). Crosssections of leaves (100 nm thick) were obtained with an EM-U-C6
ultramicrotome (Leica, Austria) and put on copper grids coated
with formvar and carbon.
TEM and X-ray microanalysis
Leaves were harvested from at least two (2–4) different batches of
plants. Samples were analyzed under a JEM 1400 transmission electron microscope (JEOL Co., Japan) equipped with a Morada CCD
camera (SiS-Olympus, Japan) at an accelerating voltage of 80 keV.
The X-ray microanalysis was performed using the energy-dispersive
full range X-ray microanalysis system EDS INCA Energy TEM
(Oxford Instruments, UK). The detection and semi-quantitative
analysis of calcium and antimony were carried out by collecting
X-ray spectra from a selected region of interest in the energy range
of 1–10 keV (Fig. 1). The relative content of calcium and antimony
was calculated using standards on the basis of the peak area characteristic of the calcium–Kα emission line (3.691 keV) and antimony–
Lα emission line (3.605 keV), with Oxford INCA TEM 200 software.
The identification and localization of calcium precipitates in cells
was determined by mapping the distribution of the element in certain parts of the tissue (X-ray mapping). The semi-quantitative analysis was carried out by measuring the spectra from identical squares
(10 µm2) for 200 s, at ×15 000 magnification, from the peripheral
areas of mesophyll cells including the cell wall and a fragment of
cytoplasm (Fig. 1). The regions inside the vacuole and intercellular space, which rarely contained visible precipitates, were chosen
as controls. The data on content of elements were processed using
EDS INCA software for the assessment of the percentage weight
concentration of elements after correction for interelement effects
(Wt%). Spectra were recorded in at least five (5–9) different regions
of the leaf mesophyll. A 3D model of precipitates was created with
a tomographic holder and Chimera software. The surface area for
precipitate cross-sections was measured with the particle analysis
procedure available in ImageJ. Particle analysis was performed on
3956 | Łabuz et al.
Fig. 1. (A) Example X-ray spectra obtained from a selected rectangular region of the periphery of an Arabidopsis wild-type mesophyll cell (including the
cell wall and a fragment of cytoplasm), which contains precipitates of calcium and antimony. The relative content (Weight %) of individual elements was
calculated on the basis of the peak area (arrow) characteristic of the calcium–Kα line (Ca K) and antimony–Lα line (Sb L). The error value quoted is sigma,
which is the statistical error for the calculated Wt%. (B) An example map showing calcium (yellow) and antimony (red) localization within the analyzed
cells. (C) Control X-ray spectra obtained from a selected rectangular region of the periphery of a mesophyll cell (including the cell wall and a fragment of
cytoplasm) not treated with potassium pyroantimonate. Chl, chloroplast; IS, intercellular space; V, vacuole.
images showing leaf mesophyll (magnifications between ×400 and
×1020), which were manually segmented into cell regions (cell wall,
vacuole, and tonoplast). The surface area of precipitates adjacent
to the outer face of the cell wall was expressed per unit of the cell
wall length, while the area of precipitates localized in the vacuole at
the tonoplast was expressed per unit of the tonoplast length. The
significance of the effects of light conditions and the plant line on
the mean calcium content and precipitate area was assessed with
ANOVA. For pairwise comparison of means, Tukey’s test was performed after one-way ANOVA, calculated separately for each plant
line. Adjusted P-values from Tukey’s test are indicated in the figures
with asterisks. The tests were performed with R-software.
Results
The KPA precipitation method does not depict the overall
distribution of calcium within cells, but only that fraction
which is susceptible to precipitation. Any calcium present in
Light-dependent calcium changes in Arabidopsis mesophyll | 3957
an appropriate concentration, not too tightly bound to cellular organelles and not readily washed out during the procedure, may be analyzed. The analysis of KPA precipitates
yields information about the difference in the distribution and
content of calcium dependent on a given factor, so the interpretation of results should be done on the basis of controls.
In our experimental model, Arabidopsis leaves were exposed
to blue light of 100 μmol m−2 s−1 for 3 min to activate phot2
and induce a measurable chloroplast avoidance response. In
wild-type plants, this time point corresponds to the moment
when chloroplasts achieve maximum velocity after the onset
of strong blue light (Łabuz et al., 2015). Control leaves were
incubated in the dark or irradiated with equimolar red light
which does not affect phototropins. Carefully selected conditions, including the use of a phosphate buffer with a slightly
alkaline pH along with glutaraldehyde fixation, were optimized for the formation of pyroantimonate precipitates only
with calcium ions, while reducing the specificity of the reaction to monovalent ions and Mg2+ (Wick and Hepler, 1982).
Spectra (Fig. 1A) and maps (Fig. 1B) of relevant cell areas
were collected in order to confirm the co-localization of calcium (marked yellow) with antimony (marked red) in these
electron-dense structures. A control spectrum showing the
element composition in cells treated without KPA is shown
in Fig. 1C.
The localization of calcium in the mesophyll cells of
wild-type Arabidopsis
In the mesophyll cells of dark-adapted Arabidopsis wild-type
leaves, calcium precipitates formed circular, semi-circular, or
lenticular structures of different sizes (Fig. 2A–D). At the cell
wall these spherical structures had several layers concentrically propagating in both directions, from and into the cell
Fig. 2. (A–D) The localization of calcium and antimony precipitates in Arabidopsis wild-type mesophyll cells in darkness. Arrows indicate precipitates of
calcium and antimony at the cell wall; the white arrowhead precipitates in the cytosol; a double arrow precipitates in the vacuole; and black arrowheads
precipitates adjacent to the chloroplast envelope and the tonoplast. CW, cell wall, Chl, chloroplast; IS, intercellular space; V, vacuole.
3958 | Łabuz et al.
(Fig. 2A, C). Precipitates of higher densities were observed
along the cell walls (Fig. 2D). Circular precipitates and very
small granules were occasionally found in the cytosol, in
the vacuole (Fig. 2C), and at the chloroplast envelope outer
membrane (Fig. 2D).
In cells irradiated with blue light for 3 min, the calcium
localization pattern was similar to that in darkness. However,
the size and density of structures in the regions of cell walls
facing the intercellular spaces were significantly increased
(Fig. 3A, D). There were relatively few precipitates in the
middle lamella (Fig. 3B). Single (Fig. 3C, F) and clustered
(Fig. 3A, D) structures were found along the edges of cell
walls. Some of these were surrounded by dark gray or black
bands of varying thickness arranged in several concentric
layers (Fig. 3C, D). A typical picture of cells after blue light
treatment showing the connected precipitates which align
along the cell wall is shown in Fig. 3A. Analysis of cell
wall cross-sections revealed that calcium precipitates were
formed by overlapping hemispheres (Fig. 3F). This observation was confirmed by a 3D model based on tomography
images. The 3D structure was limited by the thickness of
the slice (100 nm), but it demonstrated that cross-sections
indeed represent the segments of a sphere (Fig. 3D, E). In
areas where precipitates were rarely seen in dark conditions, such as the cytosol (Fig. 3B, white arrow), tonoplast,
and the chloroplast envelope (Fig. 3A), they became more
abundant after blue light treatment (Fig. 3A, B, D). Semicircular or lenticular precipitates on the tonoplast were
oriented towards the vacuole interior (Fig. 3A, long white
arrow) and those observed on the outer membrane of the
chloroplast envelope pointed towards the cytosol (Fig. 3A
short white arrow).
Fig. 3. (A, B) The localization of calcium and antimony precipitates in Arabidopsis wild-type mesophyll cells after 3 min blue light treatment of
100 μmol m−2 s−1. Cross-section of the precipitates (C, D) perpendicular and (F) parallel to the cell wall plane. (E) The 3D model of the precipitate created
from (D). Arrows indicate precipitates of calcium and antimony at the cell wall; white arrowheads precipitates in the middle lamella; and black arrowheads
precipitates adjacent to the chloroplast envelope and the tonoplast. A long white arrow indicates precipitates on the tonoplast directed towards the
vacuole interior; a short white arrow precipitates on the outer membrane of the chloroplast envelope pointing towards the cytosol; and a medium sized
arrow indicates precipitates in the cytosol. CW, cell wall, Chl, chloroplast; IS, intercellular space; V, vacuole.
Light-dependent calcium changes in Arabidopsis mesophyll | 3959
After 3 min of red light treatment, the precipitates at the
cell wall were much smaller and did not form the characteristic spherical structures (Fig. 4A–D) as compared with
darkness and blue light. No precipitates were observed in the
cytosol, on the tonoplast, and on the outer membrane of the
chloroplast envelope (Fig. 4A, B).
The localization of calcium in the mesophyll cells of
Arabidopsis phototropin mutants
In order to link the differences in Ca2+ patterns observed
after light treatments with phot2 signaling, the localization
of calcium precipitates in the phot2 and phot1phot2 mutants
was investigated. Only phot2 is responsible for calcium mobilization in mature Arabidopsis leaves under the light conditions used in this study (100 μmol m−2 s−1), as demonstrated
by Harada et al. (2003). Figure 5 shows the localization of
calcium precipitates in the cells of the phototropin mutants.
Generally, a smaller variation in calcium structures at the
cell wall was observed in these mutants. Calcium precipitates
forming spherical structures were rarely multilayered. In
dark conditions, the phot2 and phot1phot2 mutants had precipitates with a firm ‘bead’ structure, which usually merged
into bands running along the cell walls, as compared with the
Arabidopsis wild type (compare Figs 2 and 5 phot2 darkness,
phot1phot2 darkness). In the phot2 mutant, structures at the
cell wall under blue light and red light were similar to those
observed under dark conditions. Precipitates found in phot2
after red light had a defined spherical structure, in contrast
to those observed in wild-type plants (compare Figs 4 and 5
phot2 red light). Round precipitates localized in the cytosol,
at the chloroplast envelope, and the tonoplast were frequently
found in the phot2 mutant, regardless of the experimental
conditions. In the double phototropin mutant, calcium precipitates after blue light were comparable with those in darkness. After red light treatment, they were much smaller and
resembled those observed in wild-type plants.
Calcium content and the area of precipitates in
Arabidopsis mesophyll cells
To quantify the effects of light observed in the phototropin
mutants, the calcium content and the area of precipitates
were measured. The calcium content was determined semiquantitatively, based on X-ray microanalysis spectra. The
spectra from rectangular areas of the same size covered the
periphery of a mesophyll cell (including the cell wall and a
fragment of cytoplasm) lying along the cell wall (Fig. 1A).
For each spectrum on the cell periphery, two control spectra were measured in regions of the vacuole and intercellular spaces. Usually small amounts of calcium were found in
these control areas (results not shown). The lowest calcium
content at the cell wall was in wild-type plants in darkness
(Fig. 6A). Following blue light treatment, the calcium content
was considerably higher than in dark conditions. This effect
was also observed after red light, but was less prominent. In
Fig. 4. (A–D) The localization of calcium and antimony precipitates in Arabidopsis wild-type mesophyll cells after 3 min red light treatment of
100 μmol m−2 s−1. Arrows indicate precipitates of calcium and antimony at the cell wall. Chl, chloroplast; IS, intercellular space; V, vacuole.
3960 | Łabuz et al.
Fig. 5. The localization of calcium and antimony precipitates in mesophyll cells of the Arabidopsis phototropin mutants phot2 and phot1phot2 in
darkness, after 3 min of blue or red light treatment of 100 μmol m−2 s−1. Chl, chloroplast; IS, intercellular space; V, vacuole.
darkness the phot2 and phot1phot2 mutants had a higher calcium content in the analyzed regions than wild-type plants.
In the phot2 mutant, the amount of calcium increased only
slightly after blue light treatment as compared with darkness, but it increased strongly after red light irradiation. The
calcium content in the double phototropin mutant did not
change significantly after blue light, as compared with darkness. Red light treatment decreased the content of calcium
in this mutant, but this effect was not statistically significant.
Changes in the antimony content in precipitates at the cell
wall reflected those observed for calcium in all experimental
groups (Fig. 6B).
To quantify the precipitates in cell wall regions facing the
intercellular space, their surface area was calculated and normalized to the cell wall length (Fig. 7A). Only the precipitates
seen in contact with the cell wall were taken into account.
In wild-type plants the precipitate area was larger after blue
light treatment as compared with dark-adapted and red lighttreated plants. The phot2 mutant exhibited larger precipitates after red light treatment, while the difference between
darkness and blue light was insignificant. In the phototropin
double mutant, light conditions did not influence the area of
precipitates. In all light conditions and plant lines, the precipitates in the vacuole were located mainly at the tonoplast.
Their surface area was expressed per unit of tonoplast length.
In wild-type plants, the surface area was significantly higher
in blue light-irradiated than in dark-adapted leaves (Fig. 7B).
No precipitates were observed in red light-irradiated samples.
In phot2 and the double mutant, the precipitate area was not
significantly affected by light conditions.
Discussion
Calcium imaging (for a review, see Batistic and Kudla, 2012)
in mesophyll cells after light treatment presents a number
of difficulties. A major obstacle to the use of fluorescence
microscopy lies in the autofluorescence of cell components
(Miyawaki et al., 1999). The introduction of fluorescent dyes
is inefficient and results in toxic effects which hamper chloroplast movements, as shown in Lemna (Tlałka and Fricker,
1999). The cameleon system, which relies on the sensitivity
of calmodulin to calcium and on the resonance energy transfer between different green fluorescent protein (GFP) variants, is difficult to handle because the excitation wavelength
Light-dependent calcium changes in Arabidopsis mesophyll | 3961
Fig. 6. The relative content of (A) calcium and (B) antimony in precipitates at the periphery of mesophyll cells (including the cell wall and a fragment of
cytoplasm) of the Arabidopsis wild type and phot2, phot1phot2 mutants. The relative content of a given element in precipitates from dark-adapted leaves
(black bars), leaves after blue light irradiation (blue bars), and leaves after red light irradiation (red bars). Each bar shows an average of 5–9 spectra of cell
regions obtained from leaves harvested from 2–4 independent plant batches. Error bars indicate the SE. *P=0.01–0.05; **P=0.001–0.01; ***P <0.001.
Fig. 7. The surface area of precipitate cross-sections outside the cell walls (A) facing the intercellular space or (B) localized in the vacuole at the tonoplast
in wild-type, phot2, and phot1phot2 mutant Arabidopsis mesophyll cells. The area was measured in dark-adapted leaves (black bars), blue lightirradiated leaves (blue bars), and red light-irradiated leaves (red bars). Each bar shows an average of at least five images of mesophyll obtained from
leaves harvested from 2–4 independent plant batches. In (B), the bar for the wild-type red light group is missing as no precipitates at the tonoplast were
observed in these conditions. Error bars indicate the SE. *P=0.01–0.05; **P=0.001–0.01; ***P <0.001.
overlaps with the action spectra of phototropins (Miyawaki
et al., 1999). The use of the aequorin system is also limited,
as the detection method allows the measurement of calcium
changes only after light pulses (Baum et al., 1999; Harada
et al., 2003) and provides no direct image of calcium distribution in the cell. The KPA precipitation method chosen in
this work identifies a Ca2+ fraction which is relatively loosely
bound to cell components and visualizes the dynamics of
these mobile ions after a chosen stimulus (Wick and Helper,
1982). The major disadvantage of KPA precipitation is that
it does not allow changes in calcium concentration to be continuously monitored. The time resolution of the method is
limited by the speed of KPA and fixative penetration in the
tissue; thus, real-time investigation of calcium signaling during the onset of strong blue light is not possible. However,
the combination of X- ray analysis with TEM yields more
detailed information about calcium localization thanks to its
higher resolution as compared with methods based on optical microscopy. In this work, the presence of calcium precipitates within the cell walls, for example in the middle lamella
and at the border of the intercellular space or protoplast, was
shown very precisely. The morphology of precipitates was
also determined and it was confirmed that they indeed contain Ca2+. This would be virtually impossible using fluorescence techniques.
Blue light-dependent calcium localization in
Arabidopsis mesophyll cells
In wild-type Arabidopsis mesophyll cells in darkness, concentric calcium precipitates localized predominantly at the cell
wall. Some were also found in the cytoplasm. Their positioning suggests that the flow of calcium ions takes place in both
directions, both into and out of the cytoplasm. After 3 min of
blue light irradiation (100 μmol m−2 s−1), spherical structures at
the cell wall were bigger, multilayered, and aligned in clusters.
Precipitates in the cytoplasm and adjacent to the chloroplast
envelope or the tonoplast were also observed. These results
demonstrate that blue light causes specific calcium mobilization within the Arabidopsis mesophyll cells. However, the
nature of changes in loosely bound calcium content remains
obscure. The activity of membrane channels in mesophyll
protoplasts after 15 min of blue light (Stoelzle et al., 2003)
and changes in [Ca2+]cyt after a 10 s blue light pulse (Harada
3962 | Łabuz et al.
et al., 2003) were examined in Arabidopsis leaves. In continuous blue light of a considerably lower intensity (25 μmol m−2
s−1), Ca2+ fluxes were analyzed using an ion-selective microelectrode only in decapitated Arabidopsis hypocotyls. In these
conditions, the maximum calcium influx into the cell occurred
3 min after irradiation, then calcium efflux began (Babourina
et al., 2002). The increase in the number of precipitates at
the cell wall and the tonoplast may reflect Ca2+ efflux from
the cytoplasm. The alignment of spherical structures which
started to connect to each other after blue light irradiation
supports this assumption. This efflux may be a part of calcium signal dissipation, a mechanism through which the
cell sensitivity to light is sustained during constant illumination. This hypothesis is also consistent with previous results
obtained on Lemna cells stained with Fluo-3 as a calcium
indicator. Continuous, strong blue light (75.2 μmol m−2 s−1)
induced an increase in Fluo-3 fluorescence in the second minute, followed by a decrease in intensity, reaching its initial
value after 10 min (Tlałka and Fricker, 1999).
The circular precipitates and granules observed in the
cytoplasm imply the presence of vesicles rich in calcium.
As blue light induces their formation, it may be speculated
that a fraction of calcium undergoes exo/endocytosis during
phototropin signaling. Calcium in multivesicular compartments plays a signaling role during the response to pathogens (An et al., 2006). Ca2+-containing vesicles co-localized
in the proximity of the Mougeotia chloroplast. This calcium
store was suggested to be important for efficient chloroplast
rotation (Wagner and Rossbacher, 1980; Rossbacher and
Wagner, 1984). Interestingly, phot2 which is bound to the
plasma membrane moves from the cytoplasm into the Golgi
complex and post-Golgi structures after blue light treatment
(Aggarwal et al., 2014). The physiological role of this receptor trafficking remains unknown, but it might imply that phototropin is involved in formation of the calcium signature in
the cytoplasm.
After equimolar red light irradiation, calcium precipitates
at the cell wall were smaller and lacked the multilayered structure, but their calcium content measured near the cell wall
was elevated as compared with darkness. Our results are in
line with the findings of Harada et al. (2003) regarding a
red light-induced transient increase in [Ca2+]cyt in wild-type
Arabidopsis leaves.
The disturbance of calcium homeostasis in phot2 and
phot1phot2 Arabidopsis mutants
In both phototropin mutants, the effect of blue light on calcium
content present in wild-type plants, was abolished. Calcium
precipitates lacked multilayer structures. In the phot1phot2
mutant, precipitates rarely occurred adjacent to the chloroplast envelope and the tonoplast, where they were common
in wild-type cells (Fig. 7B). Semi-quantitative analysis of the
calcium content in the precipitates at the periphery of mesophyll cells confirms microscopic observations. The amount
of calcium in the examined regions in both mutants was significantly higher in darkness (Fig. 6). Also the number and
area of precipitates in the vacuole in darkness were higher in
mutants than in wild-type plants (Fig. 7B). Both lines of evidence point to disturbed calcium homeostasis in the studied
phototropin mutants and suggest that phot2 may also regulate calcium content in darkness. Phot2 can indeed actively
function in cells even in the absence of light, as shown for the
dark positioning of chloroplasts (Suetsugu et al., 2005).
In wild-type Arabidopsis mesophyll cells, blue light causes
accumulation of calcium in the examined regions. This effect
is negligible in the phot2 mutant and absent in the phot1phot2
mutant, indicating that in strong blue light calcium transport
between the symplast and apoplast depends mainly on phot2.
According to Harada et al. (2003), phot1 controls changes in
[Ca2+]cyt in leaves only in the range of 1–50 μmol m−2 s−1 of blue
light. This study confirms that blue light of 100 μmol m−2 s−1
does not affect phot1-mediated calcium signatures in
Arabidopsis mesophyll, as no calcium elevation in precipitates at the cell periphery was observed in the phot2 mutant.
Changes in calcium patterns generated after red light were
observed in wild-type and phot2 plants, but not in the double
mutant. The effect of red light on loosely bound calcium has
been previously reported by Tretyn et al. (1992), who observed
an increase in the number of KPA precipitates outside the
plasma membrane and in the ER cisternae after 5 min red
light irradiation in oat coleoptiles. This suggests that red light
irradiation leads to Ca2+ removal from the cytoplasm. In the
present study, a similar effect was observed in Arabidopsis,
but its magnitude was substantial only in the phot2 mutant.
The mechanism by which the presence of phot2 reduces the
red light effect on Ca2+ localization remains elusive. Direct
interaction between phytochromes and phototropins may
be involved, as Arabidopsis phyA and phot1 were shown to
interact at the plasma membrane (Jaedicke et al., 2012). Its
physiological relevance seems likely when considering the
effect of red light on the cytoskeletal organization observed
in the phot2 background. In wild-type Arabidopsis the organization of the cortical actin cytoskeleton is similar in the blue
and red light-irradiated mesophyll cells (Krzeszowiec et al.,
2007). In contrast, in the phot2 mutant, strong red light causes
a distinct shortening of actin filaments. Thus, phot2 together
with a red light photoreceptor has been suggested to control
F-actin organization. Phytochrome B might be this photoreceptor, as it has been proposed to attenuate the signaling
pathway leading to the chloroplast avoidance response controlled by phot2 (Luesse et al., 2010). The results presented
herein suggest the involvement of calcium in arranging the
actin cytoskeleton under red light in the phot2 mutant.
Calcium homeostasis is severely disturbed in the phot1phot2
mutant, which is reflected in the lack of light-specific changes
in calcium content at the periphery of mesophyll cells. This
confirms the involvement of phototropins in controlling calcium levels in mesophyll cells after light treatment.
Phototropin2-dependent chloroplast calcium patterns
Although precipitates adjacent to the chloroplast envelope
and to the tonoplast were observed in several experimental
groups, they were particularly abundant after blue light in
Arabidopsis wild-type cells. In the double phot1phot2 mutant,
Light-dependent calcium changes in Arabidopsis mesophyll | 3963
these structures were less frequently observed, thus their formation and localization seems to depend on the presence of
phototropins. The lack of phot2 causes a non-specific, lightindependent formation of precipitates inside the cell. This
observation is consistent with the proposed role of internal
calcium stores in the phot2 signal transduction pathway
(Harada et al., 2003). Internal Ca2+ stores are involved in the
control of chloroplast movements, as was shown by inhibitor
studies (Tlałka and Gabrys, 1993; Tlałka and Fricker, 1999;
Aggarwal et al., 2013a). It is possible that the identified calcium precipitate patterns result from a response to blue light
generated by the chloroplast. The mechanism by which the
chloroplast synchronizes the direction of movement with its
physiological state; that is, the efficiency of photosynthesis or
the photo-oxidative damage risk, is unknown. Calcium may
be a good candidate because light modulates its homeostasis
inside the chloroplast. A light-dependent calcium influx into
the chloroplast has been reported (for a review, see Johnson
et al., 2006). On the other hand, a thylakoid protein, CAS (a
Ca2+-sensing receptor) is responsible for calcium elevation in
the cytoplasm (Nomura et al., 2008), showing that the chloroplast may generate calcium signatures in the cell. In Lemna,
an increase in the level of calcium in cell wall areas neighboring with chloroplasts occurs during the chloroplast avoidance
response chemically induced by lead (Samardakiewicz et al.,
2015). In this work, almost no precipitates have been observed
inside chloroplasts. This may be due to the inaccessibility
of calcium to pyroantimonate precipitation in chloroplasts,
where calcium is mainly bound to thylakoid membranes and
stromal proteins (Stael et al., 2012).
Calcium distribution in the context of chloroplast
movements
In mesophyll cells, chloroplast movements are the main phototropin-dependent responses to blue light. Several lines of
evidence show that Ca2+ is involved in the signaling from phototropins to chloroplasts in different species (see Banaś et al.,
2012). This study shows that calcium relocalization after blue
light treatment requires the presence of phot2, as it is absent
in the phot2 mutant. This mutant also lacks full chloroplast
avoidance in strong light, since phot1 alone can trigger only
residual avoidance, followed by accumulation (Luesse et al.,
2010; Łabuz et al., 2015). Thus the observed calcium localization pattern appears to be important for eliciting the full
chloroplast avoidance response.
Acknowledgements
The study of the impact of light on calcium homeostasis was supported
by a grant from the Polish National Science Center [grant no. 2011/01/N/
NZ3/00280]. The 3D model of calcium precipitates was funded within the EU
framework of FP7, Marie Curie ITN CALIPSO [GA 2013-ITN-607-607],
and a grant from the Polish National Science Center [grant no. 2011/01/B/
NZ3/02160]. JL is a beneficiary of funding from the Jagiellonian University
within the SET project (co-financed by the European Union). The Faculty of
Biochemistry, Biophysics and Biotechnology of the Jagiellonian University
is a partner of the Leading National Research Center (KNOW) supported
by the Ministry of Science and Higher Education, and benefits from structural funds from the European Union [grant no. POIG.02.01.00-12-064/08].
The microscopic studies were performed in the Laboratory of Electron
Microscopy, Nencki Institute of Experimental Biology Warsaw, Poland
and were supported by statutory funds to this Institute. The equipment
used in this Laboratory: JEM1400 (JEOL Co., Japan) combined with EDS
INCA Energy TEM (Oxford Instruments, UK), a tomographic holder,
and a MORADA CCD camera (SiS Olympus, Germany) was financed
by EU Structural Funds, project: Centre of Advanced Technology BIM Equipment purchase for the Laboratory of Biological and Medical Imaging.
The authors would like to thank Rafał Bartosiewicz, Szymon Suski, and
Henryk Bilski for technical assistance during microscopic studies.
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